Dov Schwartz

Mutant p53 protein expression interferes with p53-independent apoptotic pathways

Loss of normal p53 function was found frequently to interfere with response of cancer cells to conventional anticancer therapies. Since more than half of all human cancers possess p53 mutations, we decided to explore the involvement of mutant p53 in drug induced apoptosis. To further evaluate the relationship between the p53-dependent and p53-independent apoptotic pathways, and to elucidate the function of mutant p53 in modulating these processes, we investigated the role of a p53 temperature-sensitive (ts) mutant in a number of apoptotic pathways induced by chemotherapeutic drugs that are currently used in cancer therapy. To that end, we studied the M1/2, myeloid p53 non-producer cells, and M1/2-derived temperature-sensitive mutant p53 expressing clones. Apoptosis caused by DNA damage induced with γ-irradiation, doxorubicin or cisplatin, was enhanced in cells expressing wild type p53 as compared to that seen in parental p53 non-producer cells; mutant p53 expressing clones were found to be more resistant to apoptosis induced by these factors. Actinomycin D, a potent inhibitor of transcription, as well as a DNA damaging agent, abrogated the restraint apoptosis mediated by mutant p53. These observations suggest that while loss of wild type p53 function clearly reduces the rate of apoptosis, p53 mutations may result in a gain of function which significantly interferes with chemotherapy induced apoptosis. Therefore, to achieve a successful cancer therapy, it is critical to consider the specific relationship between a given mutation in p53 and the chemotherapy selected.

Integrity of the N-terminal transcription domain of p53 is required for mutant p53 interference with drug-induced apoptosis.

The present study examined whether the ability of mutant p53 to block apoptosis depended on its transcriptional activity. A core domain mutant p53 (143 Val to Ala), in which two N‐terminal residues (22 and 23) essential for transactivation were also mutated (Leu to Glu and Trp to Ser, respectively), was examined. While p53 containing only the core mutation efficiently interfered with drug‐induced apoptosis, further modification at the N‐terminus abolished this blocking activity. Furthermore, expression of c‐myc, a suggested target for core mutant p53 transactivation, was elevated in the core mutant p53‐expressing cells, but was abolished in the presence of the transcription‐deficient p53 core mutant. In addition, wild‐type p53, mutated in the N‐terminus (residues 22 and 23), was unable to induce apoptosis by itself. Nevertheless, it synergized with drugs in the induction of apoptosis. This suggests that the integrity of the N‐terminus is essential for both the activity of wild‐type p53 in apoptosis and for mutant p53‐mediated block of drug‐induced apoptosis. This supports the notion that core p53 mutants act via a gain of function mechanism.

Accumulation of wild-type p53 protein upon gamma-irradiation induces a G2 arrest-dependent immunoglobulin kappa light chain gene expression.

The exposure of cells to DNA‐damaging agents leads to the accumulation of wild‐type p53 protein. Furthermore, overexpression of the wild‐type p53, mediated by transfection of p53‐coding cDNA, induced cells to undergo apoptosis or cell differentiation. In this study we found that the gamma‐irradiation that caused the accumulation of wild‐type p53 in 70Z/3 pre‐B cells induced, in addition to apoptosis, cell differentiation. This was manifested by the expression of the kappa light chain immunoglobulin gene that coincided with the accumulation of cells at the G2 phase. Overexpression of mutant p53 in 70Z/3 cells interferes with both differentiation and accumulation of cells at the G2 phase, as well as with apoptosis, which were induced by gamma‐irradiation. Furthermore, the increment in the wild‐type p53 protein level following gamma‐irradiation was disrupted in the mutant p53 overproducer‐derived cell lines. This suggests that mutant p53 may exert a dominant negative effect in all of these activities. Data presented here show that while p53‐induced apoptosis is associated with the G1 checkpoint, p53‐mediated differentiation, which may be an additional pathway to escape the fixation of genetic errors, may be associated with the G2 growth arrest phase.

Role of wild type p53 in the G2 phase: regulation of the gamma-irradiation-induced delay and DNA repair.

Up-regulation of the p53 protein was found to induce cell cyle arrest at the G1/S border and in some cases at the G2/M border. Futhermore, it was suggested that p53 is associated with the induction of the various DNA repair pathways. Previously, we demonstrated that cells coexpressing endogenous wild type p53 protein, together with dominant negative mutant p53, exhibit deregulation of apoptosis, G1 arrest and delay in G2 following γ-irradiation. IN the present study, we investigated the role of p53 protein in the DNA damage response at the G2 phase. Using p53-null, wild type p53 and mutant p53-producer cell lines, we found that the two C-terminally spliced p53 forms could prevent γ-irradiation induced muatgenesis prior to mitosis, at the G2/M checkpoint. We found that at the G2 phase, p53 may facilitate repair of DNA breaks giving rise to micronuclei, and regulate the exit from the G2 checkpoint. At the G1 phase, only the regularly spliced form of p53 caused growth arrrest. In contrast, both the regularly and the alternatively spliced p53 forms directed postmitotic micronucleated cells towards apoptosis. These results provide a functional explanation for the cell cycle-independent expression of p53 in mnormal cycling cells, as well as in cells where p53 is up-regulated, following DNA damage.

Accentuated apoptosis in normally developing p53 knockout mouse embryos following genotoxic stress.

In order to identify the alternative pathways which may substitute for the p53 function during embryogenesis, we have focused our studies on p53−/− normally developing mouse embryos that survived a genotoxic stress. We assumed that under these conditions p53-independent pathways, which physiologically control genomic stability, are enhanced. We found that while p53+/+ mouse embryos elicited, as expected, a p53-dependent apoptosis, p53−/− normally developing mice exhibited an accentuated p53-independent apoptotic response. The p53-dependent apoptosis detected in p53+/+ embryos, was an immediate reaction mostly detected in the brain, whereas the p53-independent apoptosis was a delayed reaction with a prominent pattern observed in epithelial cells of most organs in the p53-deficient mice only. These results suggest that in the absence of p53-dependent apoptosis, which is a fast response to damaged DNA, p53-independent apoptotic pathways, with slower kinetics, are turned on to secure genome stability.

Cellular events and the pattern of p53 protein expression following cyclophosphamide-initiated cell death in various organs of developing embryo.

This study was aimed at characterizing the temporal patterns of cell responses and p53 protein expression in the limbs, head, and liver of embryos responding to cyclophosphamide (CP)-induced teratogenic insult. ICR murine embryos were examined 24, 48, or 72 h after injection of 40 mg/kg CP on day 12 of pregnancy. The cellular events and temporal pattern of p53 protein expression were determined by FACS analysis and by TUNEL (apoptosis) in the head, limbs, and liver of the embryos. All tested organs showed apoptosis and a significantly decreased proportion of live cells after 24 h. Subsequent events were organ-dependent. In the liver, there were no dysmorphic events at any time and excessive cell death had been almost compensated for by 48 h. Compensation was preceded by G(1) arrest and accompanied by an increased level of p53 protein in surviving cells. Excessive cell death in the head and the limbs resulted in structural anomalies. In the head, there was an increased level of p53 protein and G(1) arrest after 24 h and the number of live cells at 48 h was equal to that seen in earlier samples, despite apoptosis. In the limbs, however, only isolated viable cells were seen by 48 h, but there was no increased level of p53 protein or G(1) arrest. Results of this study suggest that the differential sensitivity of tested organ systems to CP may be associated with differences in cellular events following CP-initiated cell death. They also suggest that the input of p53 in determining the response of these organ systems to CP-induced teratogenic insult may be different. Teratogenesis Carcinog. Mutagen. 19:353-367, 1999.

Induction of Apoptosis and p53 Expression in Immature Thymocytes by Direct Interaction with Thymic Epithelial Cells

Apoptosis of normal thymocytes was shown to be triggered by several mechanisms (e.g. glucocorticoids, γ‐irradiation). In the present study the authors report on thymocyte apoptosis that is induced by thymic epithelial cells. The thymocytes undergo a massive apoptotic death within 24 h of cocultivation with thymic epithelial cell monolayers derived from primary cultures (PTEC) or from a thymic epithelial cell line (TEC). Non‐thymic monolayers were inactive. Apoptosis induction in this experimental model requires direct contact between the thymocytes and the thymic epithelial monolayer and can be blocked by anti‐CD2 and anti‐LFA‐1 antibodies. The immature CD3−/+dull CD4+CD8+ thymocytes were the cells which undergo apoptosis. The fact that the authors are dealing with a massive apoptotic process of immature cells in the absence of exogenous antigen suggests that it involves the nonselected thymocytes. The apoptotic pathway selected by thymocytes following their culturing on TEC involves p53 expression. Indeed it was found that TEC‐induced apoptosis, led to the accumulation of p53 protein that preceded the step of DNA fragmentation in freshly isolated thymocytes as well as in a glucocorticoid resistant thymoma cell line. Since glucocorticoid‐induced thymocyte apoptosis is p53‐independent, glucocorticoids are conceivably not involved in TEC‐induced thymocyte death. The in vitro experimental model presented here may reflect the physiological sequence of events leading to thymocyte death in the thymus.

Karyotype analysis of the monogenetic trypanosomatid Leptomonas collosoma

In order to develop a genetic system for the monogenetic trypanosomatids, we have analyzed the molecular karyotype of Leptomonas collosoma based on chromosome separation by clamped homogeneous electric field (CHEF) gel electrophoresis. The chromosome location of 5 RNA coding genes (SL, U6, 5S, 7SL and rRNA) and 2 protein coding genes (for HSP83 and alpha-tubulin) was determined. All of the L. collosoma genes examined were found on at least 2 chromosomes, which differ in size in the range of 100-500 kb, suggesting that the organism is diploid. The weighted sum of L. collosoma chromosomes separated by CHEF analysis was approximately 62 +/- 3 Mb, whereas the genome size determined by FACS was estimated at approx. 80 Mb. This suggests that some of the homologous chromosomes differ in their size. The analysis presented here may facilitate studies on the function of individual genes, and on the genetic stability of this organism.